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Higher Biology
Unit 1: DNA and the Genome
1 The Structure and replication of DNA
Notes
DNA Structure
1. DNA is made up of nucleotides.
2. Each nucleotide is made up of a phosphate, a deoxyribose sugar and an organic base.
phosphate
organic base
deoxyribose sugar
3. There are 4 types of organic base – adenine, thymine, cytosine and
guanine.
adenine
ytosie
4. Nucleotides are joined to form long strands – a covalent bond forms
between the deoxyribose sugar of one nucleotide and the phosphate
of the next nucleotide.
5. DNA is made of 2 strands of nucleotides joined together by the organic bases.
6. Two polynucleotide chains that run in different directions (ANTIPARALLEL).
7. The 3’ prime end of one chain is opposite the 5’ end of the other chain
(see next page)
8. Adenine pairs with thymine and cytosine pairs with guanine.
9. The base pairs are held together by weak hydrogen bonds.
Adenine always pairs with Thymine
Cytosine always pairs with Guanine
They are “Complementary Base
Pairs”
10. The two strands are coiled round to form a double helix.
Understanding the 5’ and 3’ Ends
Organisation of DNA - Prokaryotic and Eukaryotic Cells
Prokaryotic chromosomes
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Cells that lack a membrane bound nucleus e.g bacterium
Bacteria have circular chromosomal DNA and plasmids
Prokaryotes usually have a single double-stranded and circular chromosome.
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In bacteria, the DNA is packaged tightly, along with associated proteins, into a given
area of the cell called the nucleoid.
Eukaryotic chromosomes
 Cells that do possess a membrane bound nucleus e.g animal, plant, yeast
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Eukaryotes have several linear chromosomes contained within a membrane-bound
nucleus.
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Eukaryotic cells also contain extra packages of DNA outwith the nucleus:
mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA).
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mtDNA is found in both plants and animals, whereas chloroplast DNA is only found in
green plants and certain protists.
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MtDNA is often circular, double-stranded and lacking in the structural proteins of the
nuclear chromosomes, much like the chromosomes found in prokaryotes.
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CpDNA is structured similarly to mtDNA: it is circular, double-stranded and lacks
structural proteins.
Eukaryote
DNA packaged into a
set of linear
chromosomes
Mitrochondrial
DNA located within
the cytoplasm
Chloroplast DNA in
some species
Genetic material
contained within the
nucleus
Prokaryote
Both
DNA usually found as
a double-stranded
circular molecule
DNA is packaged
into chromosomes
DNA not found within
a nucleus
The DNA is
double-stranded
Plasmids can be
present
Organisms have
a different
sequence of
bases along the
DNA
DNA is the genetic
material
DNA Replication
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Replication enables a complete copy of the genome to be passed on to each
daughter cell during mitosis.
Semi-conservative replication- each of the two resulting DNA molecules is
composed of 1 original strand and 1 new strand.
Replication of DNA requires enzymes, proteins, free nucleotides and energy (ATP).
DNA polymerase is the enzyme that joins the nucleotides in a growing DNA strand.
The strands grow in the 5’ to 3’ direction or from the 3’ to 5’ end in the growing DNA
strand.
One strand acts as a template in DNA replication.
DNA ligase is another enzyme involved in DNA replication.
DNA ligase forms Phosphodiester bonds to join DNA molecules together.
Steps for DNA Replication
1. DNA double helix unwinds
2. Weak hydrogen bonds between the bases break causing the two strands to separate
(unzip). The bases become exposed.
3. Free DNA nucleotides complementary base pair with the bases on the open strand.
4. Weak hydrogen bonds form between complementary base pairs
5. Sugar-phosphate bonds form between the nucleotides of the new strand of DNA.
Enzymes are needed for this process.
6. The DNA winds back up into the double helix.
DNA Replication
1. The DNA molecule unwinds
2. Weak __________ bonds between base pairs break allowing the two stands to
separate (‘unzip’).
3. Their bases are now exposed at a Y-shaped replication fork
4. The enzyme DNA polymerase controls the sugar phosphate of the new nucleotides
into the new DNA strand.
5. It can only add nucleotides to a pre-existing chain, i.e. where there is a primer – a
short series of nucleotides at the 3’ end of the parental DNA to be replicated.
6. Synthesis occurs in a 5’ to 3’ direction (on the new strand)
7. The DNA nucleotides will then bind to their complementary partners on the template
strand (following the base pairing rule) at the 3’ end.
8. DNA polymerase brings about the formation of the complementary sugar-phosphate
bond and the nucleotides.
9. DNA polymerase is only able to add nucleotides to the free 3’ end of a growing
strand. Therefore the DNA template strand has to be replicated in fragments (called
Okazaki fragments) each starting at the 3’ end of a primer.
10. Once the fragments are complete the primer is replaced by DNA
11. They are then joined together by an enzyme called ligase.
12. This type of fragment formation is called discontinous.
PCR
 The Polymerase Chain Reaction (PCR) is a technique that is used
widely in molecular biology.
 The process is used to amplify a single sequence of DNA into many
more identical copies, PCR can produce millions of copies from one
DNA template strand in a couple of hours.
 The name PCR is derived from a key component used in the
process, DNA polymerase, which can be used as it can work at high
temperatures which would denature many other enzymes.
 The first process in PCR involves heating to a high temperature
resulting in the DNA becoming denatured, this causes the doubled
stranded molecule to separate giving 2 single strands which act as
templates from which copies can be made.
 The second stage requires the use of oligonucleotides called DNA
primers, which are simply short sections of single stranded DNA.
The primers will find the specific nucleotide complimentary
sequence on both template strands and anneal to them giving a
starting point from which copies can be made.
 The final stage of PCR is where the short primers have DNA building
blocks called nucleotides added to them resulting in the extension
of the DNA strand. This is where the DNA polymerase does it job by
adding the nucleotides according to the sequence of the
complimentary strand, it starts at the point on the strand where the
primers have annealed in the previous step and continues to work
its way along the template.
 This process occurs to both single template strands simultaneously
so from one section of original DNA 2 identical copies will be made.
 The 3 stages are referred to as a cycle. Each cycle will double the
number of DNA template strands.
 This results in 2 copies of DNA being produced after the 1st cycle, 4
copies produced in the 2nd cycle, 8 copies in the 3rd cycle , etc so
by the time the 30th cycle has completed there will be
1,073,741,824 identical copies of DNA! PCR can be applied to many
forensic and medical techniques used widely today.
PCR Process
1.
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4.
5.
6.
It is possible to identify particular short sequences of base pairs (bp) on a
DNA strand by first amplifying the target sequence using the technique
called polymerase chain reaction (PCR).
PCR can amplify any DNA sequence hundreds of millions of times in just a
few hours. It is especially useful because it is highly specific, easily
automated and capable of amplifying minute amounts of sample.
The whole process is only possible because of a special heat-stable
enzyme called Taq polymerase, isolated from thermophilic bacteria.
Tac polymerase is needed because the process of DNA Polymerase
requires single stranded DNA fragments to be produced by heating to
95C
The enzyme Tac polymerase is able to tolerate temperatures of 95C and
has a temperature optimum of 72C.
This enzyme can synthesise the complementary strand of a given DNA
strand in a mixture containing the four DNA nucleotide bases and two
short DNA fragments called primers. Each primer is usually about 20 base
pairs (bp) long. The primers are designed to bind to the DNA at either side
of the target sequence.
Why is PCR used?
What is the point of PCR?
 PCR can be used to amplify a desired DNA sequence of any origin (virus, bacteria,
plant or animal) millions of times in a matter of hours.
Why is PCR useful?
 it is highly specific
 it is easily automated
 it is capable of amplifying minute amounts of sample.
What are Primers?
Primers are short DNA sequences or oligonucleotides (each about 20 bases long), that bind
at either side of the target sequence, one on each of the complementary strands of the
target
What is special about Taq Polymerase?
 PCR depends on special heat-stable DNA polymerase enzymes from thermophilic
bacteria, which can withstand temperatures of 95ºC and have an optimal
temperature for activity of 72ºC.
 Taq polymerase is a special DNA polymerase - it is an enzyme that is not
denatured by heating at 90oC to 95oC
Uses of PCR
 Degraded DNA samples can be amplified from some unusual sources
 Diagnosis of AIDS
 PCR has proven to be a quick, reliable method for detecting all types of mutations
associated with genetic disease
 Forensic cases
 Paternity tests
. The following questions are taken from recent past papers, and refer to PCR.
a
b.
c.